Downloaded from http://jcp.bmj.com/ on May 16, 2015 - Published by group.bmj.com
Original article
Short-term and long-term storage stability of heparin plasma ammonia Lora Dukic, Ana-Maria Simundic ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2014-202693). University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia Correspondence to Lora Dukic, University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Vinogradska c 29, Zagreb 10 000, Croatia; [email protected] Received 25 September 2014 Revised 3 December 2014 Accepted 15 December 2014 Published Online First 19 January 2015
ABSTRACT Aims Ammonia is an extremely unstable analyte and requires special attention during sampling, transport and storage. The aim of this study was to evaluate the stability of ammonia in lithium-heparin plasma during short-term (at +4°C) and long-term (at −20°C) storage. Methods Twenty plasma samples were used for shortterm stability assessment. Each sample was divided into five aliquots and stored in stoppered tubes at +4°C, for 1, 2, 3, 4 and 24 h from initial testing. Fifteen plasma samples were used for long-term stability assessment. Each sample was divided into eight aliquots and stored in stoppered tubes at −20°C for 3, 24, 48 h and 1, 2, 4, 8 and 12 weeks from initial testing. Ammonia concentration was determined on a Beckman Coulter AU2700 chemistry analyser using Randox ammonia enzymatic UV method. Bias was calculated from initial value for each time point and compared with quality specifications defined by Royal College of Pathologists of Australasia. Results The average bias exceeded the total allowable error after storage of samples for 1 h at +4°C and 3 h at −20°C. Conclusion Ammonia is not stable during storage at +4°C and −20°C in lithium-heparinised plasma and should therefore be analysed immediately.
INTRODUCTION
To cite: Dukic L, Simundic A-M. J Clin Pathol 2015;68:288–291. 288
Ammonia determination is clinically significant in various pathological states such as in patients with symptoms of neuromuscular and cerebral disturbances caused by hepatopathy, oncology patients receiving aggressive chemotherapy and patients taking the antiepileptic drug valproic acid.1 2 In neonates and children, ammonia is often requested when metabolic disturbance is suspected.2 Guidelines for detection and management of hyperammonaemia suggest that levels of ammonia up to 200 μmol/L are associated with acquired conditions such as sepsis, chemotherapy or liver dysfunction, while ammonia levels higher than 200 μmol/L indicate metabolic disorders.3 Ammonia is an extremely unstable analyte and requires special attention during sampling, transport and storage. It has been recommended that heparinised or EDTA plasma samples be kept in an ice bath after sampling, centrifuged immediately on arrival at the laboratory, aliquoted and analysed within 15–30 min, for accurate analysis of ammonia.4 Guidelines for the investigation of hyperammonaemia even recommend repeated ammonia sampling to exclude possible artefactual cause of high ammonia values.3 Unfortunately, ammonia testing is not universally available and delayed analysis due to prolonged sample transport
time may sometimes be carried out. However, existing original reports about short-term and longterm ammonia stability are inconsistent. According to the manufacturer of our assay (Randox Laboratories Ltd, Crumlin, UK), if plasma is separated within 30 min, ammonia is stable for 2 h at +2 to +8°C.5 Some authors even recommend long-term plasma storage at −70°C.6 The aim of our study was to verify the manufacturer’s declaration by assessing the stability of ammonia in lithium-heparin plasma. Stability was assessed during standard storage conditions in our laboratory for short-term (at +4°C) and long-term (at −20°C) sample storage.
MATERIALS AND METHODS From December 2013 until May 2014, routine patient samples for which ammonia testing was requested were collected. Sampling was performed in 4.5 mL Lithium Heparin plasma tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) in a single venipuncture without stasis. On sampling, whole blood was transported on ice immediately to the biochemistry laboratory. On delivery to the laboratory, the specimen was centrifuged for 10 min at 1800g in a benchtop centrifuge (Rotofix 32, Hettich, Tuttlingen, Germany). Specimens with visible haemolysis, icteria and lipaemia were not included in the study. Plasma was immediately separated from cells and 300 μL aliquot was processed for ammonia analysis on a Beckman Coulter AU2700 chemistry analyser (Beckman Coulter, Tokyo, Japan) using Randox ammonia enzymatic UV method (Randox Laboratories Ltd, Crumlin, UK). Daily quality control was performed using Ammonia/Ethanol Control Level 1 and 2 (Randox Laboratories Ltd, Crumlin, UK). Within-run precision coefficients of variation declared by the manufacturer were 3.6%, 2.5% and 2.8% for the ammonia concentrations 57.2, 167.0 and 305.0 μmol/L, respectively. Our own laboratory data for precision on patient samples are: within-run precision 14.4% and 3.7% at concentrations 24.3 and 102.5 μmol/L, respectively. Following initial testing, remaining plasma was immediately divided in several portions of 300 μL, according to the protocol described below. Twenty plasma samples were used for short-term stability assessment. Each sample was divided in five aliquots and stored in stoppered tubes at +4°C, for 1, 2, 3, 4 and 24 h from initial testing. Fifteen plasma samples were used for long-term stability assessment. Each sample was divided in eight aliquots and stored in stoppered tubes at −20°C for 3, 24, 48 h and 1, 2, 4, 8 and 12 weeks from initial testing. After thawing, plasma
Dukic L, et al. J Clin Pathol 2015;68:288–291. doi:10.1136/jclinpath-2014-202693
Downloaded from http://jcp.bmj.com/ on May 16, 2015 - Published by group.bmj.com
Original article 36.3 (IQR 28.5–67.8) μmol/L. For plasma samples used for longterm stability assessment (at −20°C; N=15), the median of initial concentration was 18.7 (IQR 16.9–25.4) μmol/L. Figure 1 and table 1 show the median ammonia concentrations and respective bias (absolute and relative) in samples stored at +4°C during different time points. After 1 h of plasma storage at +4°C, observed bias had already exceeded RCPA criteria for total allowable error (CI for estimated bias exceeded the limit of acceptance). There was a gradual increase in ammonia concentration during the first 4 h of storage, followed by almost twofold increase of initial concentration of ammonia after 24 h of storage. Median concentrations between serial measurements in samples stored at +4°C differed significantly (p
Original article
Short-term and long-term storage stability of heparin plasma ammonia Lora Dukic, Ana-Maria Simundic ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2014-202693). University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia Correspondence to Lora Dukic, University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Vinogradska c 29, Zagreb 10 000, Croatia; [email protected] Received 25 September 2014 Revised 3 December 2014 Accepted 15 December 2014 Published Online First 19 January 2015
ABSTRACT Aims Ammonia is an extremely unstable analyte and requires special attention during sampling, transport and storage. The aim of this study was to evaluate the stability of ammonia in lithium-heparin plasma during short-term (at +4°C) and long-term (at −20°C) storage. Methods Twenty plasma samples were used for shortterm stability assessment. Each sample was divided into five aliquots and stored in stoppered tubes at +4°C, for 1, 2, 3, 4 and 24 h from initial testing. Fifteen plasma samples were used for long-term stability assessment. Each sample was divided into eight aliquots and stored in stoppered tubes at −20°C for 3, 24, 48 h and 1, 2, 4, 8 and 12 weeks from initial testing. Ammonia concentration was determined on a Beckman Coulter AU2700 chemistry analyser using Randox ammonia enzymatic UV method. Bias was calculated from initial value for each time point and compared with quality specifications defined by Royal College of Pathologists of Australasia. Results The average bias exceeded the total allowable error after storage of samples for 1 h at +4°C and 3 h at −20°C. Conclusion Ammonia is not stable during storage at +4°C and −20°C in lithium-heparinised plasma and should therefore be analysed immediately.
INTRODUCTION
To cite: Dukic L, Simundic A-M. J Clin Pathol 2015;68:288–291. 288
Ammonia determination is clinically significant in various pathological states such as in patients with symptoms of neuromuscular and cerebral disturbances caused by hepatopathy, oncology patients receiving aggressive chemotherapy and patients taking the antiepileptic drug valproic acid.1 2 In neonates and children, ammonia is often requested when metabolic disturbance is suspected.2 Guidelines for detection and management of hyperammonaemia suggest that levels of ammonia up to 200 μmol/L are associated with acquired conditions such as sepsis, chemotherapy or liver dysfunction, while ammonia levels higher than 200 μmol/L indicate metabolic disorders.3 Ammonia is an extremely unstable analyte and requires special attention during sampling, transport and storage. It has been recommended that heparinised or EDTA plasma samples be kept in an ice bath after sampling, centrifuged immediately on arrival at the laboratory, aliquoted and analysed within 15–30 min, for accurate analysis of ammonia.4 Guidelines for the investigation of hyperammonaemia even recommend repeated ammonia sampling to exclude possible artefactual cause of high ammonia values.3 Unfortunately, ammonia testing is not universally available and delayed analysis due to prolonged sample transport
time may sometimes be carried out. However, existing original reports about short-term and longterm ammonia stability are inconsistent. According to the manufacturer of our assay (Randox Laboratories Ltd, Crumlin, UK), if plasma is separated within 30 min, ammonia is stable for 2 h at +2 to +8°C.5 Some authors even recommend long-term plasma storage at −70°C.6 The aim of our study was to verify the manufacturer’s declaration by assessing the stability of ammonia in lithium-heparin plasma. Stability was assessed during standard storage conditions in our laboratory for short-term (at +4°C) and long-term (at −20°C) sample storage.
MATERIALS AND METHODS From December 2013 until May 2014, routine patient samples for which ammonia testing was requested were collected. Sampling was performed in 4.5 mL Lithium Heparin plasma tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) in a single venipuncture without stasis. On sampling, whole blood was transported on ice immediately to the biochemistry laboratory. On delivery to the laboratory, the specimen was centrifuged for 10 min at 1800g in a benchtop centrifuge (Rotofix 32, Hettich, Tuttlingen, Germany). Specimens with visible haemolysis, icteria and lipaemia were not included in the study. Plasma was immediately separated from cells and 300 μL aliquot was processed for ammonia analysis on a Beckman Coulter AU2700 chemistry analyser (Beckman Coulter, Tokyo, Japan) using Randox ammonia enzymatic UV method (Randox Laboratories Ltd, Crumlin, UK). Daily quality control was performed using Ammonia/Ethanol Control Level 1 and 2 (Randox Laboratories Ltd, Crumlin, UK). Within-run precision coefficients of variation declared by the manufacturer were 3.6%, 2.5% and 2.8% for the ammonia concentrations 57.2, 167.0 and 305.0 μmol/L, respectively. Our own laboratory data for precision on patient samples are: within-run precision 14.4% and 3.7% at concentrations 24.3 and 102.5 μmol/L, respectively. Following initial testing, remaining plasma was immediately divided in several portions of 300 μL, according to the protocol described below. Twenty plasma samples were used for short-term stability assessment. Each sample was divided in five aliquots and stored in stoppered tubes at +4°C, for 1, 2, 3, 4 and 24 h from initial testing. Fifteen plasma samples were used for long-term stability assessment. Each sample was divided in eight aliquots and stored in stoppered tubes at −20°C for 3, 24, 48 h and 1, 2, 4, 8 and 12 weeks from initial testing. After thawing, plasma
Dukic L, et al. J Clin Pathol 2015;68:288–291. doi:10.1136/jclinpath-2014-202693
Downloaded from http://jcp.bmj.com/ on May 16, 2015 - Published by group.bmj.com
Original article 36.3 (IQR 28.5–67.8) μmol/L. For plasma samples used for longterm stability assessment (at −20°C; N=15), the median of initial concentration was 18.7 (IQR 16.9–25.4) μmol/L. Figure 1 and table 1 show the median ammonia concentrations and respective bias (absolute and relative) in samples stored at +4°C during different time points. After 1 h of plasma storage at +4°C, observed bias had already exceeded RCPA criteria for total allowable error (CI for estimated bias exceeded the limit of acceptance). There was a gradual increase in ammonia concentration during the first 4 h of storage, followed by almost twofold increase of initial concentration of ammonia after 24 h of storage. Median concentrations between serial measurements in samples stored at +4°C differed significantly (p
